Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells

Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in NSCs induced the immortalization of astroglial-like cells, which were then able to generate tumors with characteristics of the mesenchymal subtype of GBM on transplantation, consistent with a potential astroglial origin for mesenchymal GBM. Sequence analysis of transposon insertion sites from tumors and immortalized cells identified more than 200 frequently mutated genes, including human GBM-associated genes, such as Met and Nf1, and made it possible to discriminate between genes that function during astroglial immortalization vs. later stages of tumor development. We also functionally validated five GBM candidate genes using a previously undescribed high-throughput method. Finally, we show that even clonally related tumors derived from the same immortalized line have acquired distinct combinations of genetic alterations during tumor development, suggesting that tumor formation in this model system involves competition among genetically variant cells, which is similar to the Darwinian evolutionary processes now thought to generate many human cancers. This mutagenesis strategy is faster and simpler than conventional transposon screens and can potentially be applied to any tissue stem/progenitor cells that can be grown and differentiated in vitro.Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer in adults. Patients with GBM have a uniformly poor prognosis, with a mean survival of 1 y (1). Thus, advances on all fronts, both basic and applied, are needed to combat this deadly disease better. Recent studies have provided evidence for self-renewing, stem-like cells within human gliomas (2). These glioma-initiating cells constitute a small minority of neoplastic cells within a tumor and are defined operationally by their ability to seed new tumors (3). To target these rare glioma-initiating cells, a better understanding of the molecular mechanisms that regulate their formation is essential.Considerable progress has been made in understanding the mutations responsible for GBM. The Cancer Genome Atlas network has cataloged the recurrent genomic abnormalities in GBM by genome-wide DNA copy number events and sequence-based mutation detection for 601 genes (4). Gene expression-based molecular classification has also defined four subtypes of GBM termed proneural, neural, classical, and mesenchymal (5). Proneural GBM is enriched for the oligodendrocyte gene signature, whereas the classical group is associated with the astrocytic signature. The neural class is enriched for genes differentially expressed by neurons, whereas the mesenchymal class is associated with the cultured astroglial signature (5). Several recurrent mutations, such as PDGFRA, IDH1, EGFR, and NF1, also correlate with these GBM subtypes, providing additional support for their existence. Numerous other, often rare, mutations have also been identified in GBM. Although these datasets are valuable for understanding the molecular pathogenesis of GBM, it is still difficult to distinguish between mutations that contributed to tumor initiation and those acquired later during tumor progression.The cell of origin (COO) of GBM is still controversial. Neural stem cells (NSCs) are good candidates because the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for gliomagenesis. NSCs are also more susceptible to malignant transformation than differentiated cells in the adult brain (6, 7). However, the genetic pathways that can transform NSCs into glioma-initiating cells still remain elusive. Transposon-based mutagenesis provides an unbiased, high-throughput method for identifying genes important for GBM (8). Here, we describe a unique two-step insertional mutagenesis strategy that makes it possible to identify genes and signaling pathways that are able to transform a NSC into a cancer-initiating cell for the mesenchymal subtype of GBM. In this two-step approach, NSCs are first mutagenized in vitro and the mutagenized cells are then transplanted into immunocompromised mice for subsequent tumor development following additional rounds of transposon-based mutagenesis. This makes it possible to discriminate between the genetic changes that occur early in tumor initiation and those required for tumor progression. In addition to identifying several previously undescribed GBM candidate cancer genes, our studies suggest that transposon-induced tumors mimic the evolutionary processes now thought to generate many human cancers, in which tumors have a branched cellular and genetic architecture reminiscent of Darwin’s iconic evolutionary tree.     

Plastic-adherent progenitor cells in human bone marrow

Human bone marrow contains plastic-adherent hemopoietic progenitor cells whose plating efficiency is increased by brief (2 h) exposure to methylprednisolone (MP). When subsequently covered with methylcellulose medium, they form colonies of monoblastoid cells. Colony size, but not number, and mature cell production are increased by erythropoietin (epo) and granulocyte-macrophage colony-stimulating factor (GM-CSF). However, colonies do not grow under serum-free conditions. The resistance of plastic-adherent progenitors to treatment with 5-fluorouracil (5FU), their growth pattern, and their capacity to produce granulocytic and erythroid colonies on replating, suggest that they may be similar to the primitive, 5FU-resistant, plastic-adherent progenitor cells (HPP-CFC) in murine marrow.     

Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice

Stem/progenitor cells from bone marrow and other sources have been shown to repair injured tissues by differentiating into tissue-specific phenotypes, by secreting chemokines, and, in part, by cell fusion. Here we prepared the stem/progenitor cells from human bone marrow (MSCs) and implanted athem into the dentate gyrus of the hippocampus of immunodeficient mice. The implanted human MSCs markedly increased the proliferation of endogenous neural stem cells that expressed the stem cell marker Sox2. Labeling of the mice with BrdUrd demonstrated that, 7 days after implantation of the human MSCs, BrdUrd-labeled endogenous cells migrated throughout the dorsal hippocampus (positive for doublecortin) and expressed markers for astrocytes and for neural or oligodendrocyte progenitors. Subpopulations of BrdUrd-labeled cells exhibited short cytoplasmic processes immunoreactive for nerve growth factor and VEGF. By 30 days after implantation, the newly generated cells expressed markers for more mature neurons and astrocytes. Also, subpopulations of BrdUrd-labeled cells exhibited elaborate processes immunoreactive for ciliary neurotrophic factor, neurotrophin-4/5, nerve growth factor, or VEGF. Therefore, implantation of human MSCs stimulated proliferation, migration, and differentiation of the endogenous neural stem cells that survived as differentiated neural cells. The results provide a paradigm to explain recent observations in which MSCs or related stem/progenitor cells were found to produce improvements in disease models even though a limited number of the cells engrafted.     

SSEA-4 identifies mesenchymal stem cells from bone marrow

Adult bone marrow (BM) contains hematopoietic stem cells (HSCs) as well as a nonhematopoietic, stromal cell population. Within this stromal population are mesenchymal stem cells (MSCs), which not only support hematopoiesis but also differentiate into multiple lineages, including fat, bone, and cartilage. Because of this multipotentiality, the MSC is an attractive candidate for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, research progress has been hampered by the limited existing knowledge of the biology of these cells, particularly by the lack of a suitable marker for their prospective isolation. Here, we report that SSEA-4, an early embryonic glycolipid antigen commonly used as a marker for undifferentiated pluripotent human embryonic stem cells and cleavage to blastocyst stage embryos, also identifies the adult mesenchymal stem cell population.     

Functional tissue-engineered blood vessels from bone marrow progenitor cells

OBJECTIVE: Stem cells have significant potential for development of cell-based therapeutics for cardiovascular tissue regeneration. METHODS: We developed a novel method for isolating smooth muscle cells (SMC) from ovine bone marrow using a tissue-specific promoter and fluorescence-activated cell sorting. RESULTS: As compared to vascular SMC, bone marrow-derived smooth muscle progenitor cells (BM-SMPC) exhibited similar morphology, showed higher proliferation potential and expressed several SMC markers including alpha-actin, calponin, myosin heavy chain, smoothelin, caldesmon and SM22. When embedded in fibrin hydrogels, BM-SMPC contracted the matrix and displayed receptor- and non-receptor-mediated contractility, indicating that BM-SMPC can generate force in response to vasoreactive agonists. We also prepared tissue-engineered blood vessels from BM-SMPC and BM-derived endothelial cells and implanted them into the jugular veins of lambs. As early as five weeks post-implantation, grafted tissues displayed a confluent endothelial layer overlaying the medial layer in which BM-SMPC were aligned circumferentially and synthesized significant amounts of collagen. In contrast to previous results with vascular SMC, BM-SMPC synthesized high amounts of elastin that was organized in a fibrillar network very similar to that of native vessels. CONCLUSIONS: Our results suggest that BM-SMPC may be useful in studying SMC differentiation and have high potential for development of cell therapies for the treatment of cardiovascular disease.     

Human bone marrow progenitor cells stimulated by cord blood

Agar cultures of human bone marrow cells stimulated by cord plasma or irradiated cord blood feeder layers demonstrated the presence of a multilineage hemopoietic growth factor in cord blood. When bone marrow cultures stimulated with giant cell tumor-conditioned medium (GCT-CM) were supplemented with this cord blood-derived growth factor, total colony numbers increased by more than 50% after day 23 and persisted in culture for approximately 40 days. Marrow cultures stimulated by the cord blood-derived growth factor formed colonies of neutrophils, monocyte-macrophages, eosinophils, mast cells, and a few colonies containing a mixed cell population. The results suggest that, while GCT-CM contains granulocyte-monocyte colony-stimulating factor (GM-CSF), cord blood contains a high concentration of a multilineage hemopoietic growth factor, which may be multi-CSF.     

Selective secretion of chemoattractants for haemopoietic progenitor cells by bone marrow endothelial cells: a possible role in homing of haemopoietic progenitor cells to bone marrow

To elucidate the mechanisms by which haemopoietic progenitor cells lodge in the bone marrow, we examined the secretion of chemoattractants for haemopoietic progenitor cells by bone marrow and lung endothelial cells. The bone marrow endothelial cells, but not lung endothelial cells, secreted chemoattractants for the haemopoietic progenitor cell line, FDCP-2, and normal haemopoietic progenitor cells. Checkerboard analysis demonstrated that the conditioned medium of the bone marrow endothelial cells had chemotactic activity and random motility-stimulating activity. The bone marrow endothelial cells expressed stromal-cell-derived factor-1 (SDF-1) mRNA and produced SDF-1 protein, whereas the lung endothelial cells did not. Adhesion of FDCP-2 cells to the bone marrow endothelial cells was partially inhibited by anti-SDF-1 antibody. These findings suggest that the chemoattractants for haemopoietic progenitor cells including SDF-1 and random motility-stimulating factor(s) selectively secreted by the bone marrow endothelial cells may contribute to the homing of haemopoietic progenitor cells to bone marrow.     

Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells

OBJECTIVE: There is an increased interest in rheumatology in mesenchymal progenitor/stem cells (MPCs) and their roles in rheumatic diseases, but little is known about the phenotype of these cells in vivo. The aim of this study was to isolate and characterize human bone marrow (BM) MPCs. METHODS: Fluorescence microscopy was used to identify putative MPCs among adherent BM cells. To purify them, a positive selection with antifibroblast microbeads was used, combined with fluorescence-activated cell sorting (FACS) for microbead+,CD45(low) cells. A more detailed phenotype of these cells was determined using 4-color flow cytometry, and standard chondrogenic, osteogenic, and adipogenic assays were used to investigate their differentiation potentials. RESULTS: Putative MPCs microscopically identified as large, fibroblast-like, D7-FIB+ cells were purified using positive selection with D7-FIB-conjugated (antifibroblast) microbeads followed by FACS for specifically bound microbead+,CD45(low) cells. These cells represented 0.01% of mononuclear cells in the BM. They were uniformly positive for CD105, LNGFR, HLA-DR, CD10, CD13, CD90, STRO-1, and bone morphogenetic protein receptor type IA (BMPRIA) and were negative for CD14, CD34, CD117, and CD133. Only cells with this phenotype could proliferate and produce adherent cell monolayers capable of chondrogenic, osteogenic, and adipogenic differentiation. D7-FIB- cells in the BM lacked any MPC activity. Uncultured skin fibroblasts had a phenotype similar to that of BM MPCs, but were negative for LNGFR, STRO-1, HLA-DR, and BMPRIA. CONCLUSION: This study shows the distinct phenotype, morphology, and method of isolation of BM MPCs. The findings may have implications for defining the physiologic roles of MPCs in arthritis, bone diseases, and joint regeneration.     

Phenotypic characterization of human bone marrow granulocyte-macrophage forming progenitor cells

Cell surface antigens of the human bone marrow CFU-C have been studied. Human marrow cells were incubated with a variety of monoclonal antisera and complement prior to culture in semisolid media. By using indirect immunofluorescent studies, the percentage of bone marrow cells binding the antibodies was determined. The CFU-C phenotype is HLA+, la+, 4F2+, 3A1-, and DUALL-1-. This study provides information that is useful in the study of myeloid cell ontogeny and necessary for the use of some of these reagents in the treatment of bone marrow cells prior to human bone marrow transplantation in various clinical settings.     

Enrichment of pluripotent hemopoietic progenitor cells from human bone marrow

Pluripotent hemopoietic progenitor cells (CFU-GEMM, cells forming mixed hemopoietic colonies in methylcellulose) from human bone marrow were enriched 90-fold by positive selection on the fluorescence-activated cell sorter using monoclonal antibody RFB-1. Bone marrow cells were separated by cell size, using log 90 degrees light scatter, and the cell fraction containing CFU-GEMM was further separated by relative fluorescence intensity for the RFB-1 antigen. Further enrichment, up to 150-fold, was achieved by depleting bone marrow of T cells and mature myeloid cells prior to RFB-1 selection. These procedures yield a cell fraction containing 51% blast cells, 2% promyelocytes, and 47% undifferentiated (lymphocyte-like) mononuclear cells, although only 1% of the cells formed a mixed colony. CFU-GEMM are strongly positive for the RFB-1 antigen, whereas morphologically identifiable erythroblasts, myeloblasts, and promyelocytes are weakly RFB-1+. This suggests that the relative concentration of the RFB-1 antigen on bone marrow cells is inversely related to their maturity. The greatly increased recovery of CFU-GEMM after the separation of bone marrow by log 90 degrees light scatter and the removal of T cells and mature myeloid cells suggested that accessory cells that normally regulate the cloning efficiency of CFU-GEMM were removed.     

骨髓CD34^＋干细胞向内皮细胞转化的诱导方法

目的探讨骨髓CD34^＋细胞向血管内皮细胞转分化的诱导方法。方法采集犬骨髓,经免疫磁珠分离出内皮祖细胞,内皮细胞生长因子（VEGF）诱导分化为内皮细胞并扩增,倒置相差显微镜、免疫细胞化学和摄取DilAc—LDL试验鉴定。将所得细胞种植于人工血管,扫描电镜观察细胞形态,并与MNCs作对比。结果经流式细胞仪测定,分离后的细胞中CD34^＋细胞占78．46％±6．37％;CD34^＋细胞培养2周后细胞基本铺满培养瓶底面,细胞呈“鹅卵石”状排列,CD34^＋和Ⅷ因子免疫细胞化学染色均为阳性。扫描电镜下观察可见内皮细胞平铺于人工血管表面,有伪足伸出并长入血管内表面微孔内。结论通过免疫磁珠方法可分离得到高纯度的骨髓CD34^＋细胞,经体外培养VEGF诱导后可定向分化为内皮细胞。     

Increase in circulating bone marrow progenitor cells after myocardial infarction

BACKGROUND: Most circulating blood cells expressing the marker CD34 are bone marrow progenitor cells. These cells differentiate into cardiomyocytes, endothelial and smooth muscle cells after myocardial infarction in vivo. Mobilization of bone marrow progenitor cells into the peripheral blood after myocardial infarction may supply these cells to the heart. Rise in CD34+ cell concentrations following myocardial infarction would support the existence of myocardial-initiated mobilization. METHODS: Serial measurements of circulating CD34+ cells were made in 42 consecutive patients presenting with first ST-elevation myocardial infarction. Measurement of serum concentrations of monocyte chemoattractant protein-1, stromal derived factor-1, hepatocyte growth factor, interleukin-17 and thrombopoietin was also performed. Samples were drawn on day 1 after myocardial infarction, and on days 4, 8 and 12. Levels of CD34+ cells and cytokines were also measured in 15 controls. RESULTS: By day 8, the mean concentration of CD34+ cells rose by 74% above mean control level of 2527 cells/ml, and 41% above day 1 mean (P=0.02). This rise was sustained on day 12 (P=0.05). On day 1, there was a 9.3-fold rise in hepatocyte growth factor above the control level of 589 pg/ml (P=0.002). Hepatocyte growth factor levels declined from the day 1 mean of 6061 to 1485 pg/ml on day 12 (P=0.002). No significant change in stromal derived factor-1, interleukin-17, monocyte chemoattractant protein-1 and thrombopoietin was observed. Elevations in CD34+ cells and hepatocyte growth factor were not related to infarction size as estimated on echocardiography. CONCLUSIONS: Elevation in the concentration of circulating CD34+ cells after myocardial infarction suggests that myocardial initiated bone marrow progenitor cell mobilization exists in humans. The cytokines studied in our protocol are not likely to play a direct role in bone marrow progenitor cell mobilization.     

Hemopoietic progenitor cells and bone marrow stromal cells in patients with autoimmune hepatitis type 1 and primary biliary cirrhosis

BACKGROUND/AIMS: Autologous hematopoietic stem cell transplantation has been used in severe cases of autoimmunity. We investigated whether hemopoietic progenitor cells and/or bone marrow (BM) microenvironment are affected in autoimmune hepatitis type-1 (AIH-1) and primary biliary cirrhosis (PBC). METHODS: We studied 13 AIH-1 patients, 13 PBC patients, 12 cirrhotic controls (CC) and ten healthy controls (HC). Flow cytometry, expansion cultures, long-term BM cultures and clonogenic progenitor cell assays were used. Stromal cell function was assessed in long-term BM cultures recharged with normal CD34+ cells. RESULTS: AIH-1 had increased CD34+, CD34+/CD38+ and CD34+/CD38- cells compared to all groups (P<0.001). PBC had lower progenitor cells compared to controls (P<0.005). No differences were found between CC and HC. Committed progenitor cells in non-adherent cell fraction were increased in AIH-1 (P<0.05) but decreased in PBC compared to controls (P<0.05). Granulocyte-macrophage colony forming units (CFU) and erythroid-burst CFU were increased in AIH-1 compared to all groups (P<0.001). PBC had these CFUs decreased compared to controls (P<0.005). Stromal cells failed to support normal hemopoiesis in PBC. CONCLUSIONS: We demonstrated for the first time that AIH-1 had increased hemopoietic progenitor cells and normal stromal function. In PBC, progenitor cells and BM microenvironment were defective. Further studies will determine the significance of these novel findings.     

Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia

Cyclic neutropenia (CN) is a congenital hematopoietic disordercharacterized by remarkably regular oscillations of blood neutrophils from near normal to extremely low levels at 21-day intervals. Recurringepisodes of severe neutropenia lead to repetitive and sometimeslife-threatening infections. To investigate the cellular mechanism ofCN, the ultrastructure and the proliferative and survivalcharacteristics of bone marrow-derived CD34⁺ earlyprogenitors, CD33⁺/CD34 myeloid progenitors,and CD15⁺ neutrophil precursors from CN patients andhealthy volunteers were studied. The ultrastructural studies showedprofound apoptotic features in bone marrow progenitor cells in CN.Colony-forming assays demonstrated a 75% decrease in the number ofearly myeloid-committed colonies compared with controls. Long-termculture-initiating cell assays demonstrated a 6-fold increase inproduction of primitive progenitor cells in CN. To determine whetheraccelerated apoptosis might account for the underproduction of myeloidprogenitors, the hematopoietic subpopulations were labeled withfluorescein isothiocyanate-annexin V and analyzed by flow cytometry.Short-term culture of CN cells resulted in apoptosis of approximately65% of CD34⁺ cells, 80% ofCD33⁺/CD34 cells, and more than 70% ofCD15⁺ cells, as compared with 20%, 7%, and 15% apoptosisin respective control subpopulations. Evidence of accelerated apoptosisof bone marrow progenitor cells was observed in all 8 patientsparticipating in the study, regardless of the stage in the CN cycle inwhich bone marrow aspirations were obtained. Granulocytecolony-stimulating factor therapy of CN patients significantly improvedsurvival of bone marrow progenitor cells. These data indicate thatineffective production of neutrophils is due to accelerated apoptosisof bone marrow myeloid progenitor cells in CN.     

大鼠骨髓单个核细胞体外诱导向内皮分化的研究

目的 :为组织工程研究作准备 ,分离大鼠骨髓单个核细胞并诱导培养向内皮分化。方法 :成年SD大鼠 ,应用Ficoll淋巴细胞分离液 (密度1 .0 77g/ml) ,将长骨骨髓经非连续密度梯度离心 ,收集中层的单个核细胞 ,加入诱导培养基并置于纤维连接素包被的培养板上进行诱导分化 ,观察细胞生长状况 ,以透射电镜及Ⅷ因子、CD3 1、Lectin免疫组化以及流式细胞仪方法对培养细胞进行鉴定。结果 :细胞圆形、纺锤形单层融合贴壁生长 ;Ⅷ因子、CD3 1、Lectin免疫组化染色阳性 ;透射电镜显示细胞具有内皮特征性的Weibel Palade小体。结论 :采用Ficoll密度梯度离心可获得较高纯度的骨髓单个核细胞 ,经体外培养并诱导分化 ,具有血管内皮细胞的特征     

Detection of primitive macrophage progenitor cells in mouse bone marrow.

A previously undetected population of macrophage progenitor cells with high proliferative potential (HPP-CFC; an average of 5 x 10(4) cells/colony) in nutrient agar cultures has been demonstrated in post-fluorouracil (FU) and fluorouracil plus endotoxin (FUEt) treated and normal mouse bone marrow, using a combination of colony-stimulating factors, pregnant uterus extract (PMUE) plus human spleen-conditioned medium (HUSPCM). Neither PMUE nor HUSPCM alone stimulated colony formation by the HPP-CFC. The incidences of HPP-CFC were 1 in 2380 nucleated cells in normal marrow, 1 in 380 for 10-day post-FU, and 1 in 118 in 8-day post-FUEt marrow cells. HPP-CFC were only depleted to 57% of normal at 2 days after FU treatment, whereas the cells responsive to PMUE alone (low proliferative potential, LPP-CFC) were depleted to 1.2%, indicating a marked difference in cycling status of the respective types of progenitor cells.     

犬骨髓成年多能祖细胞诱导分化血管内皮细胞的实验研究

目的内皮细胞移植对损伤组织的修复治疗至关重要,本研究旨在探讨骨髓成年多能干细胞(MAPCs)体内外诱导分化血管内皮细胞的可行性.方法采用Percoll密度梯度法分离培养MAPCs.应用10 ng/ml血管内皮细胞生长因子(VEGF)对骨髓MAPCs进行体外诱导分化2～3周,使其定向分化成为血管内皮细胞.采用细胞形态学、细胞免疫组化以确定诱导分化的效果.将标记BrdU的MAPCs自体移植于犬心肌内,观察局部微环境对于骨髓MAPCs的分化能力.结果应用10 ng/ml VEGF孵育骨髓MAPCs 2～3周,可见MAPCs分化为血管内皮细胞:细胞形态呈鹅卵石样;形成血管样结构;细胞vWF免疫染色阳性.移植于心肌内的MAPCs在体内形成新生血管,血管内皮BrdU染色与vWF染色均阳性.结论骨髓MAPCs可在体外VEGF诱导下或在体内微环境作用下分化为成熟血管内皮细胞.骨髓MAPCs可为损伤组织的移植修复治疗提供内皮细胞资源.